Aerobic fermentation method

ABSTRACT

The present invention describes a method of culturing a micro-organism under aerobic conditions in a fermentation vessel comprising the injection of a first oxygen-containing gas into the lower portion of the vessel in a heterogeneous flow causing a chaotic motion of the broth and the introduction of a second oxygen-containing gas in the vessel characterised by introducing the second oxygen-containing gas as a heterogeneous flow of gas bubbles moving in the vessel in all possible directions, independent of the direction of the flow of the broth resulting in turbulent flow conditions at the site of injection; and as a set of gas bubbles of non-uniform size and a wide size distribution.

[0001] The present invention relates to a method of culturing amicro-organism under aerobic conditions in a fermentation vessel.

[0002] Industrial scale fermentation processes are carried out for theproduction of various products such as biomass (e.g. baker's yeast),enzymes, amino acids and secondary metabolites (e.g. antibiotics). Mostof these fermentation processes involve the culturing of micro-organismsincluding bacteria, yeasts and fungi and require the supply of oxygenfor the aerobic metabolism of these micro-organisms. Usually, the oxygenis supplied by passing an oxygen-containing gas, such as air, throughthe liquid in the fermentation vessel. The oxygen is transferred fromthe gas bubbles to the liquid phase thus allowing its uptake by themicro-organism. In fermentation processes involving large vessel volumesand high biomass densities, the transfer of oxygen from the gas to theliquid phase may become the growth limiting factor. Obtaining increasedoxygen transfer is therefore one of the targets to obtain increasedgrowth thus making these processes more economically attractive.Alternatively, fermentation processes that require for instance thecarbon- or nitrogen source as the limiting factor in order to promoteproduction of a certain product, require a non-growth limiting oxygentransfer rate. Hereto, processes have been developed that increase theoxygen transfer rate. Methods that are known in the art for increasingthe oxygen transfer rate comprise: increasing the mixing (e.g. stirring)of the liquid, increasing the flow of oxygen-containing gas and/orincreasing the oxygen concentration of the oxygen-containing gas (e.g.oxygen enriched air). Other methods are described that focus on thesupply of two separate oxygen-containing gas streams.

[0003] European Patent Application EP-A-0,222,529 discloses theprinciple of improved oxygen enrichment of a fermentation broth by usinga second oxygen-containing gas stream. The fermenting system consists ofa vessel equipped with a riser and a downcorner; air is supplied to thebroth in the riser, while in the downcorner a second oxygen containinggas is supplied to the broth. In the Japanese patent applicationJP-63-283570 this and other fermenting systems are disclosed that areequipped with means to circulate the flow, such as a draft tube or a(set of) impeller(s), an air stream for the purpose of circulation andCO₂ stripping and an oxygen supplying device that supplies oxygen in thedirection that is opposite to the stream of the circulating fluid. Thedisadvantage of such a system is that the flow of the liquid inside thevessel must be known and sufficiently stable and controlled accuratelyin order to place the oxygen supplying device in such a way that thedesired oxygen transfer is obtained. European Patent ApplicationEP-A-0,341,878 discloses that the second oxygen containing gas may besupplied to the broth in a circulation loop outside the fermenter.

[0004] European Patent Application EP-A-0477818 discloses a similarmethod involving the injection of a feed air stream into a mixing vesselprovided with impeller means having a vertical axis and separatelyinjecting additional oxygen from an additional oxygen injection point.However, this method is limited by the fact that the latter injectionpoint must be located remote from the vicinity of the air injectionpoint in order to minimize mixing of the additional oxygen with the airbubbles.

[0005] In European Patent Application EP-A-0829534, the same principleis applied to a gas driven fermentation system, i.e. a system withoutmechanical mixing devices. The system employs injecting a first oxygencontaining gas, such as air, in a set of bubbles upwardly through avessel in a heterogeneous flow causing an upward flow of the broth inthe vessel. The second oxygen-containing gas that is injected in thelower portion of the vessel is a set of bubbles also moving upwardlythrough said vessel in a homogeneous flow. The homogeneous flow isdefined by the inventors as that the flow has a uniform gas bubbledistribution and a narrow bubble size distribution wherein there is noobservable gas/liquid downflow. The homogeneous character of this secondgas is stated to be an essential feature. In contrast to the disclosureof JP-62-119690, the second oxygen-containing gas is not provided intothe broth within the fermentor vessel at a region where the broth isflowing downwardly, rather, it must be provided into the broth where itis rising.

[0006] In European Patent Application EP-A-0,901,812 the method isfurther improved by providing oxygen directly into a ‘stationaryvortex’, a rotating body of liquid with little or no axial or transversemovements supposedly created by a mechanical agitation system. Theadvantage is said to maintain the oxygen bubbles within the vortex untilthe oxygen bubble have dissolved into the reaction mixture. However, inindustrial vessels it will be very difficult to locate the preciseposition of these stationary regions. Moreover, in highly turbulentgas-liquid mixtures as often applied in industry, it is likely that thevortices are not at all stationary.

[0007]FIG. 1. Small bubble size distribution for different distancesfrom the sparger.

[0008]FIG. 2. Large bubble size distribution for different distancesfrom the sparger

[0009] A homogeneous flow is defined herein as a flow in which allbubbles rise with the same velocity and the mixture follows straightstreamlines with the absence of recirculatory liquid flows. Homogeneousflow can occur only when sparger holes are evenly distributed at thebottom of the vessel, and at superficial gas velocities <approx. 0.04m/s.

[0010] A heterogeneous flow is defined herein as a flow in which localdifferences in liquid velocity will occur with the presence ofrecirculatory liquid flows. Heterogeneous flow will occur when spargerholes are unevenly distributed at the bottom of the vessel, or, atsuperficial gas velocities >approx. 0.04 m/s when sparger holes areevenly distributed.

[0011] Chaotic motion of the broth is defined herein as a movement,characterised by a direction and a velocity, which has a dependence onits history and conditions in the vessel and which is bound by certainlimits (e.g. velocities can never exceed a certain maximum). This isdifferent from random motion, which is a statistical quantity which hasno dependence on the history, and is not bound by limits (e.g. extremelyhigh velocities can occur, though with extremely low chance)

[0012] Turbulent flow is defined herein as liquid movement in which themomentum differences of individual liquid elements cannot be damped outby the viscosity of the liquid. As a result, circulating eddies will beformed in which liquid velocity differences will become more intense,with vigorous motion of the liquid.

[0013] A uniform size distribution of gas bubbles is defined herein asbeing a symmetrical distribution with a single maximum.

[0014] A non-uniform size distribution of gas bubbles is defined hereinas a non-symmetrical distribution (e.g. with a tail to one side) or adistribution with more than one maximum.

[0015] A narrow size distribution of gas bubbles is defined herein as adistribution in which >95% of the bubbles have a diameter falling withinthe interval between 0.2*Ø_(b) and 5*Ø_(b), where Ø_(b) is the averagebubble diameter.

[0016] A wide size distribution of gas bubbles is defined herein as adistribution in which >95% of the bubbles have a diameter falling withinthe interval between 0.01*Ø_(b) and 100*Ø_(b), where Ø_(b) is theaverage bubble diameter.

[0017] The present invention provides a method of culturing amicro-organism under aerobic conditions in a fermentation vesselcomprising the injection of a first oxygen-containing gas into the lowerportion of the vessel in a heterogeneous flow causing a chaotic motionof the broth and the introduction of a second oxygen-containing gas inthe vessel characterised in that the second oxygen-containing gas isintroduced as a heterogeneous flow of gas bubbles in the vessel in allpossible directions and independent of the direction of the flow of thebroth resulting in turbulent flow-conditions at the site of injectionand as a set of gas bubbles of non-uniform size and a wide sizedistribution. The first oxygen-containing gas may contain from 15-30%oxygen (v/v), preferably from 20-30% oxygen and most preferably is air.The first oxygen-containing gas is injected into the lower portion ofthe fermentation vessel, preferably near the bottom. The secondoxygen-containing gas may contain from 30-100% oxygen, preferably from70-100% oxygen and most preferably is from 90-100% oxygen. The injectionpoint of the second oxygen-containing gas is not limited to a certainarea of the fermentation vessel and can be both in the vicinity orremote from the injection point of the first oxygen-containing gas,irrespective of whether the fermentation vessel is equipped with amechanical stirring device or not. The advantage of the method of thepresent invention is that the oxygen transfer obtained by this method isimproved over the existing methods of the prior art and does not put anylimitations to the inlet position of the second oxygen-containing gasstream, thus allowing more flexibility in the fermentation vesseldesign. The method of the present invention is suitable for culturingyeasts, fungi and bacteria. Preferred examples of yeasts are members ofthe genera Saccharomyces or Kluyveromyces such as Saccharomycescerevisiae and Kluyveromyces lactis. Preferred examples of fungi aremembers of secondary metabolite producing genera such as Penicillium(e.g. Penicillium chrysogenum for the production of penicillin and otherantibiotics) and Acremonium and members of enzyme producing genera suchas Aspergillus and Trichoderma. Preferred examples of bacteria aremembers of the genera Streptomyces, Escherichia, Pseudomonas orBacillus.

[0018] One preferred embodiment is a method for culturing amicro-organism under aerobic conditions in a fermentation vessel whereinthe broth is mixed by mechanical stirring. In another preferredembodiment, no mechanical stirring is applied but mixing of the broth isobtained by the first-containing gas stream such as air, i.e. the bellcolumn fermenter.

[0019] The second-oxygen containing gas can be introduced in thefermentation vessel by means of simple pipes or gas spargers, orifices,venturi type nozzles, gas-liquid nozzles or supersonic gas injectionnozzles. Preferably, the second-oxygen containing gas is introduced byone or more nozzles each comprising at least one hole. More preferably,the second-oxygen containing gas is injected in the fermentation vesselin three dimensions. This can be obtained by using several nozzles eachcomprising at least one hole which nozzles are positioned in thefermenter in a suitable arrangement to give injection in threedimensions. Alternatively, one or more nozzles may be used eachcontaining at least 3 holes arranged such to give injection in threedimensions.

EXAMPLE 1

[0020] Air was sparged through a nozzle fitted in the vicinity of thebottom of a glass tank (0.6 m diameter) filled with 300 liter of a 4%Na₂SO₄ solution in water. The Na₂SO₄-solution was used to mimic thecoalescence properties of a fermentation broth and to provide thetransparency needed for the detection techniques that were used. Thenozzle contained 7 holes of 1 mm diameter, 6 of which were equallyspaced in a circle of 32 mm diameter and one positioned in the center.The nozzle flow was directed upward. Gas hold-up and bubble sizedistribution were measured in the gas plume at various verticalpositions above the sparger. The gas hold-up was measured with aradioactive transmission technique using a Cs137 gamma radiation sourceand a Nal detector. This technique measures the total density of thegas-liquid mixture in the detection zone vessel and thus allows thecalculation of the gas fraction. The bubble size distribution expressedas Sauter mean d32 was determined by image analysis of video recordingsof optical magnifications of the bubble patterns (T. Martin—PhD thesis:Gas dispersion with radial and hydrofoil impellers in fluids withdifferent coalescence characteristics. 1996. Herbert Utz Verlag, München(D)). Table 1 and FIGS. 1 and 2 show the results for a nozzle exitvelocity of 490 m/s. A non-uniform bubble size distribution was observedwith typical peaks in the small range between 0.2 and 0.3 mm (FIG. 1)and in the large diameter range between 2 and 4 mm (FIG. 2) with atypical holdup of 1.1% for the small bubbles and 1-3% for the largebubbles. TABLE 1 Vertical Gas hold-up Sauter d32 position Small Largerbubbles Small bubbles Larger bubbles (cm) bubbles (%) (%) (mm) (mm) 51.1 0.1 0.267 2.273 14 1.1 1.3 0.216 2.630 41 1.1 2.2 0.287 3.587

EXAMPLE 2

[0021]Saccharamyces cerevisiae was cultured in a bubble column fermenterequipped with an air (i.e. the first oxygen containing gas) sparger nearthe bottom of the fermenter and a recirculation loop with an externalheat-exchanger for cooling. Superficial air flow rates wereapproximately 0.20 m/s and the oxygen transfer was 0.4-0.6% per meter ofthe unaerated broth height. The broth was circulated through the heatexchanger four times per hour. Other fermentation conditions were asdescribed by Reed, G and Nagodawithana, T. W. in chapter 6 of YEASTTECHNOLOGY (1991, Van Nostrand Reinhold, New York). The second oxygencontaining gas consisted of pure oxygen and was injected below the airsparger, employing nozzles operated under a pressure of approximately 5bar with hole diameters in the order of 1 mm, in order to achievesupersonic injection velocities of the oxygen, resulting in anon-uniform oxygen bubble distribution. We selected the inlet positionbelow the oxygen sparger to allow the fraction of larger oxygen bubbles(50-70 vol %) to move upward and coalesce with the air bubbles and toallow the fraction of small bubbles to be entrained in the recirculationflow. In this way, optimum pressure and residence time conditions werecreated to achieve near total oxygen uptake (100% efficiency), whereasthe fraction of large bubbles showed the transfer efficiency of the airflow. The ratio of the two gas flows (air/oxygen) was 9:1. Yeastproductivity was approximately 8-9 kg biomass (dry wt) per kg broth perhour which is twice the productivity found without injection of thesecond oxygen-containing gas. The following results are depicted inTable 2. TABLE 2 O₂ Transfer O₂ Transfer Rate Relative Efficiency (mmoleO₂ per kg O₂-Transfer Rate O₂-containing gas (%) broth per hour) (-fold)1^(st) Air 20 140 1 2^(nd) Pure O₂ large bubbles 20 60 small bubbles 10070 Total: 270 1.9

EXAMPLE 3

[0022] A fermentation was carried out similar to the one described inExample 2 except that the ratio of the two gas flows (air/oxygen) was6:1 and the yeast productivity was approximately 16 kg biomass (dry wt)per kg broth per hour. Table 3 gives the results. TABLE 3 O₂TransferO₂Transfer Rate Relative Efficiency (mmole O₂ per kg O₂-Transfer RateO₂-containing gas (%) broth per hour) (-fold) 1^(st) Air 15 140 1 2^(nd)Pure O₂ large bubbles 15 90 small bubbles 100 270 Total: 500 3.6

[0023] Compared to Example 2, a further increase in the O₂-Transfer Ratewas observed which is explained by the fact that a higher O₂ consumptionoccurred as a result of an increased productivity of the yeast.

EXAMPLE 3

[0024]Saccharomyces cerevisiae was grown in a stirred tank fermenter,equipped with an air sparger below the bottom impeller, and a secondsparger which was placed in the radial liquid flow stream generated bythe bottom impeller. The impeller was of the turbine disk type. Thesecond sparger was equipped with nozzles of the type as described inexample 2, and through this sparger a second oxygen containing gas wassupplied, consisting of pure oxygen. The position of the second spargerwas chosen to be located in the most vigorously agitated part of theliquid as to demonstrate that the positive effect of the generation ofthe oxygen bubbles is not limited to locations where the flow conditionsare relatively stagnant.

[0025] In addition to a reference fermentation with only air, twoexperiments have been executed with extra oxygen. In one fermentation,the air stream was enriched by replacing approximately 6% of the air bypure oxygen. In the second fermentation, the same amount of oxygen wassupplied by direct injection through the second sparger, while havingthe air flow in the first sparger reduced to 94%. The oxygenconcentration in the liquid phase was controlled at 20% of airsaturation. Results of the oxygen transfer rate at the end of thefermentation are shown in Table 4. TABLE 4 O₂ transfer rate Experiment(mmole O₂ per kg broth per hour) Reference 35 Enrichment 44 Directinjection 50

[0026] The improvement of the oxygen transfer rate with the enriched aircan be attributed to a higher driving force from gas to liquid phase.The measured magnitude is in agreement with the expectations.

[0027] The improvement with the direct injection experiment can beattributed to a cumulative contribution of transfer by the air from theair sparger, by a fraction of large oxygen bubbles from the secondsparger and a fraction of small bubbles also from the second sparger.Assuming that the ratio of large to small oxygen bubbles is 5:1, thenthe contributions of air, large oxygen bubbles and small oxygen bubblesto the total-oxygen transfer rate is 34, 10 and 6 mmoles per kg brothper hour, respectively.

1. A method of culturing a micro-organism under aerobic conditions in a fermentation vessel comprising the injection of a first oxygen-containing gas into the lower portion of the vessel in a heterogeneous flow causing a chaotic motion of the broth and the introduction of a second oxygen-containing gas in the vessel characterised by introducing the second oxygen-containing gas as a heterogeneous flow of gas bubbles moving in the vessel in all possible directions, independent of the direction of the flow of the broth resulting in turbulent flow conditions at the site of injection; and as a set of gas bubbles of non-uniform size and a wide size distribution.
 2. A method according to claim 1 wherein the micro-organism is a yeast, fungus or bacterium.
 3. A method according to claim 2 wherein the yeast belongs to the genus Saccharomyces or Kluyveromyces.
 4. A method according to claim 3 wherein the yeast is Saccharomyces cerevisiae
 5. A method according to claim 3 wherein the yeast is Kluyveromyces lactis.
 6. A method according to claim 2 wherein the fungus is a member of the genera Penicillium, Acremonium, Aspergillus or Trichoderma
 7. A method according to claim 6 wherein the fungus is Penicillium chrysogenum.
 8. A method according to claim 2 wherein the bacterium is a member of the genera Streptomyces, Escherichia, Pseudomonas or Bacillus.
 9. A method according to anyone of the preceding claims wherein the broth is mixed by mechanical stirring.
 10. A method according to anyone of the preceding claims wherein the second-oxygen containing gas is introduced in the fermentation vessel by means of one or more nozzles each comprising at least one hole.
 11. A method according to claim 10 wherein the nozzles are positioned in the fermentation vessel so as to obtain injection of the second-oxygen containing gas in three dimensions.
 12. A method according to claim 10 wherein the holes are positioned in the nozzle so as to obtain injection of the second-oxygen containing gas in three dimensions. 